About thirty cell surface proteins have been shown to use a covalently attached glycosyl-phosphatidylinositol (GPI) molecule for membrane anchoring. It has been suggested that this novel anchoring structure is the site for degradation by specific phospholipases leading to the release of the protein from the cell surface and the generation of molecules with second messenger activity. Variations in the structure of the GPI anchor might have a profound influence on the ability of the protein either to be released by endogenous GPI anchor-specific phospholipases C and D or to act as a source of active second messengers. This hypothesis will be tested using mammalian alkaline phosphatase (APase) as a model system. APase will be released from placenta and intestine by phosphatidylinositol-specific phospholipase C (PI-PLC) and purified. The C-terminal glycopeptides will be isolated and their composition determined by GC and GC/MS. The neutral glycans prepared from these glycopeptides will be analysed for structural variations by HPLC and high resolution gel filtration. Similar studies will be done using biosynthetically labelled APase prepared from cells in culture in order to determine if the observed structural variations are cell type specific. The-molecular mechanism of resistance of APase to PI-PLC that has been observed previously will be investigated. Anchoring of the PI-PLC resistant APase by a modified GPI anchor or a transmembrane polypeptide will be determined by biosynthetic labelling and peptide antibodies against the C-terminus. The functional significance of variations in GPI anchor structure will be assessed. The sensitivity of APase to purified endogenous GPI-specific phospholipases C and D will be determined. The effect of variations in composition and structure on the ability of the inositol-glycan moiety in the released APase to modulate the activity of insulin-sensitive enzymes will also be measured. The results of these studies will increase our understanding of the important physiological role played by these novel biological structures.